Human cytomegalovirus (HCMV) is a clinically important herpesvirus that has coevolved for millions of years with its human host, and establishes lifelong persistent infection. A substantial proportion of its 235 kb genome is dedicated to manipulating host immunity through targeting antiviral host proteins for degradation or relocalization. Quantitative proteomics of the infected cell has extensively characterized these processes, but the cell-free virion has been less well studied. We therefore carried out proteomic analysis of a clinical HCMV strain (Merlin) virion. This revealed 18 novel components, including the viral protein gpUL141, which is recognized as an NK immune-evasin that targets several host proteins (CD155, CD112, and TRAILR) when expressed within the cell. Coimmunoprecipitation of gpUL141 from virions identified interactions with viral entry glycoproteins from the trimer (gH/gL/gO), pentamer (gH/gL/UL128/UL130/UL131A), and gH/gpUL116 complexes, as well as gB. Only interactions with gH/gB occurred in the absence of other viral proteins. Analysis supported a model in which gpUL141 homodimers independently interacted with separate gB/gH-containing complexes. gpUL141 encodes an ER retention domain that restricts trafficking through the ER/Golgi, and limited the transport of glycoprotein complexes bound by gpUL141. As a result, gpUL141 reduced levels of multiple glycoprotein complexes on the infected cell surface as well as in the virion. This reduced syncytium formation, inhibited antibody-dependent cellular cytotoxicity (ADCC), and reduced susceptibility to neutralizing antibodies. Thus, gpUL141 represents an immune-evasin that not only targets host proteins to limit NK-cell attack, but also alters the trafficking of multiple viral glycoprotein complexes in order to evade humoral immunity.

Human cytomegalovirus (HCMV) is a clinically important herpesvirus that has co-evolved for millions of years with its human host, and establishes lifelong persistent infection. A substantial proportion of its 235kb genome is dedicated to manipulating host immunity through targeting antiviral host proteins for degradation or relocalisation. Quantitative proteomics of the infected cell has extensively characterised these processes, but the cell-free virion has been less well studied. We therefore carried out proteomic analysis of a clinical HCMV strain virion. This revealed multiple novel components, including the viral protein gpUL141, which is recognised as an NK immune-evasin that targets several host proteins (CD155, CD112, and TRAILR) when expressed in the cell. Co-Immunoprecipitation of gpUL141 from virions identified interactions with viral entry glycoproteins from the trimer (gH/gL/gO), pentamer (gH/gL/UL128/UL130/UL131A), and gH/gpUL116 complexes, as well as gB. These interactions occurred via direct interactions between gpUL141 and either gH or gB. Analysis supported a model in which gpUL141 homodimers independently interacted with separate gB/gH-containing complexes. gpUL141 encodes an ER retention domain that restricts trafficking through the ER/golgi, and limited the transport of glycoprotein complexes bound by gpUL141. As a result, gpUL141 reduced levels of multiple glycoprotein complexes on the infected cell surface as well as in the virion. This reduced syncytia formation, inhibited antibody-dependent cellular cytotoxicity (ADCC), and reduced susceptibility to neutralising antibodies. Thus, gpUL141 represents an immune-evasin that not only targets host proteins to limit NK-cell attack, but also alters the trafficking of multiple viral glycoprotein complexes in order to evade humoral immunity. ### Competing Interest Statement The authors have declared no competing interest.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

This chapter reviews the development of some of the most powerful mammalian vector systems currently available derived from three virus families: the adenoviruses, alphaviruses and poxviruses. The three groups of viruses are unrelated but their associated vectors share a capacity to provide for high-level expression in vitro and particular emphasis will also be given to their application to vaccination and immunotherapy. Since smallpox eradication vaccinia virus and other poxvirus-based vectors have actively developed to generate a popular range of vectors with enhanced safety and efficacy. For vaccination, there are many examples where a single immunization with a recombinant virus can elicit effective, long-lived protection. Though the authors will concentrate on aspects and applications of recombinant vaccinia virus (rVV), due to conservation of the expression machinery within the poxvirus family, many of the principles of rVV can be applied to other poxviruses.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Human cytomegalovirus (HCMV) is one of the most widespread, highly successful herpesviruses, establishing a life-long viral infection in humans. HCMV has been described as a paradigm of immune evasion able to manipulate many immune functions in the host. Here, we describe a novel, post-translational mechanism in which HCMV downregulates a disintegrin and metalloproteinase 17 (ADAM17), a ‘sheddase’ that cleaves and releases over 80 membrane-anchored cytokines, cell adhesion molecules and other receptors. A screen of HCMV deletion mutants identified UL148 and UL148D as the HCMV genes responsible for ADAM17 downregulation, working synergistically to alter ADAM17 levels in infected cells. We demonstrate that UL148/UL148D interfere with ADAM17 maturation, resulting in expression of only the intracellular immature precursor, and absence of mature ADAM17 on the surface of wildtype HCMV-infected cells. The consequences of ADAM17 downregulation by HCMV were analysed using proteomics and validated using biochemical and flow cytometric techniques, revealing impact on multiple cell surface and secreted host proteins. This included stabilisation of surface TNF Receptor 2, as well as Vasorin and Jagged1, which have recognised roles in Treg development. Other known ADAM17 targets were not stabilised, suggesting specific control by HCMV. Vaccinia virus, another paradigm of immune evasion, also impaired surface ADAM17 expression, suggesting that manipulation of ADAM17 may represent a novel immunoregulatory hub targeted by large DNA viruses.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Human cytomegalovirus (HCMV) extensively modulates host cells, downregulating >900 human proteins during viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a mass spectrometry-based interactome analysis of 169 tagged, stably-expressed canonical strain Merlin HCMV proteins, and two non-canonical HCMV proteins, in infected cells. This identified a network of >3400 virus-host and >150 virus-virus protein interactions, providing insights into functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to SH3 domains of NCK Adaptor Protein-1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection.

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

SignificanceHuman cytomegalovirus (HCMV) is the major infectious cause of developmental disorders in babies due to its capacity to cross the placenta. HCMV is also a major pathogen in transplant recipients and HIV–AIDS patients. Despite inducing the strongest immune responses observed for any human pathogen, HCMV evades host defenses and persists for life. Herein, we report another viral stealth strategy. HCMV UL148 reduces surface expression of a key cell adhesion molecule (CD58), impairing the ability of NK and T cells to be activated by HCMV-infected cells. Our findings highlight a role for CD58 in recognition of HCMV-infected cells and may be relevant for development of future antiviral therapies.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. https://doi.org/10.7554/eLife.22206.001 eLife digest Cytomegalovirus (CMV) is one of eight herpesviruses that can infect humans. Most people will at some point become infected with CMV, yet the virus tends only to cause serious disease in people whose immune system is not working properly. Individuals living with HIV/AIDS and organ transplant recipients (who have to take drugs that suppress their immune system to prevent the organ being rejected) are particularly vulnerable to CMV infections. Critically, the virus can cross the placenta to infect of the foetus. CMV infection in the womb can cause miscarriage, lead to severe developmental problems in babies and is a major cause of deafness. Herpesvirus infections are for life. While the immune system cannot eliminate CMV, it does have many systems that combine to sense and control infections. Natural killer cells are known to play a critical role in detecting and destroying cells infected with CMV. The virus, in turn, has nine genes that help to protect it against natural killer cells. This includes two genes that belong to a group of similar genes called the US12 family, but it is not clear whether other members of this gene family also provide protection against natural killer cells. Fielding et al. now show that at least four members of the US12 gene family help CMV to evade natural killer cells. For example, two members work together to target a human protein called B7-H6 that acts a sensor to alert natural killer cells if a particular cell is infected. However, the impact of the US12 family goes even wider. The whole family works together to control proteins that are found on the surface of human cells, and many of these proteins appear to be involved in regulating the immune response. The findings of Fielding et al. provide an insight into how the US12 gene family works, and how CMV has evolved to escape the human immune system. New therapies to control CMV infections are urgently needed so the next challenge is to design new antiviral agents that will target CMV’s defence systems. https://doi.org/10.7554/eLife.22206.002 Introduction At 236 kb the human cytomegalovirus (HCMV) genome is the largest of any characterized human virus and is comprised of long and short unique regions (UL and US), each flanked by inverted terminal repeats. HCMV codes for around of 170 canonical protein-coding genes with 39 herpesvirus ‘core’ genes concentrated in the center of the UL region (Dolan et al., 2004). The core genes mainly encode structural components of the virion and proteins required for virus DNA replication and have orthologues in the other human herpesviruses. The vast majority of the remaining HCMV genes are not essential for virus replication in vitro (Dunn et al., 2003) yet are replete with accessory functions, many of which have been implicated in suppressing host immune responses. Unusually, HCMV encodes 15 gene families of variable size that are often clustered on the genome (Davison et al., 2002; Holzerlandt et al., 2002; Chee et al., 1990; Dolan et al., 2004; Davison et al., 2003). Many of these gene families exhibit homology with cellular genes and are conserved to various extents in other primate CMVs. Consequently, these primate CMV gene families are likely to have arisen through gene capture and amplification driven by differential selective pressures in their various primate hosts over millennia (Davison et al., 2013, 2003). The US12 gene family consists of 10 genes, designated US12 to US21, arranged sequentially in the US region and transcribed in the same orientation (Chee et al., 1990; Dolan et al., 2004). The genetic arrangement of the US12 family is reminiscent of ‘accordion’ gene expansions, which are generated when a cellular or virus resistance function is placed under strong selective pressure (Filée, 2013). Such an expansion was recently exemplified experimentally using a poxvirus interferon resistance function (Elde et al., 2012). The US12 family encodes a series of 7-transmembrane spanning proteins with low-level homology to the cellular transmembrane bax-inhibitor one motif-containing proteins (TMBIM). While not essential for virus replication, the US12 family has been implicated in HCMV tropism, virion maturation and immune evasion (Das and Pellett, 2007; Cavaletto et al., 2015; Bronzini et al., 2012; Hai et al., 2006; Gurczynski et al., 2014; Fielding et al., 2014). Natural Killer (NK) cells play a critical role in controlling HCMV infections, and the virus invests a substantial proportion of its coding capacity to inhibit NK cell activation (Wilkinson et al., 2013). We previously observed that US18 and US20 suppress cell surface expression of the NK cell-activating ligand MICA (Fielding et al., 2014) and posited that the synergistic action of US18 and US20 may be the vestige of an immune selective pressure that drove the original expansion of the US12 family. These data show that multiple US12 family members can co-operate to target the same cellular protein. Therefore individual functions, as identified with single gene viral mutants, may not be readily replicated by expressing these same viral genes in isolation, i.e. these viral genes may work more efficiently in the context of HCMV productive infection. To investigate the function of all US12 family genes, we undertook a systematic functional analysis that showed four members were NK immunevasins. Conventional biochemical investigations on US12 family proteins are rendered problematic due to their extreme hydrophobicity. We therefore undertook multiplexed Tandem Mass Tag (TMT)-based proteomic analyses to systematically evaluate the capacity of all US12 family genes to modulate the cellular proteome, both individually and in concert. Such an approach has been enabled by our recent development of Plasma Membrane Profiling (PMP) to identify novel cell surface targets for the HCMV latency protein UL138 (Weekes et al., 2013) and individual viral immunevasins UL141 and US2 (Hsu et al., 2015). Quantitative Temporal Viromics (QTV) allowed >8000 cellular and 153 viral proteins to be tracked throughout the course of productive HCMV infection, thus building a comprehensive picture of cellular control by the virus (Weekes et al., 2014). Through comparative analysis of ~1300 cell surface and ~7200 cellular proteins during infection with HCMV US12 family deletion mutants, we now describe in detail how this family has a profound influence not only on NK cell recognition but other key functions that impact on immunity including cellular adhesion and cytokine signalling. Results US12 family members differentially affect NK cell activation To determine whether the US12 family has a broader role in modulating NK cell responses, a series of HCMV US12 family deletion mutants were generated (10 single deletion mutants and the US12-21 ‘block’ deletion). The HCMV genome was manipulated by DNA recombineering in a strain Merlin BAC that did not express RL13 and UL128 (Stanton et al., 2010). Viruses were generated by DNA transfection and the complete genomic sequence of the virus stocks was validated by deep sequencing (Table 1). In NK functional assays, the △US12-21 block deletion mutant induced substantially higher levels of NK cell degranulation compared to the parent HCMV with all four different donors tested (Figure 1B). Significantly increased levels of NK activation were detected in assays using deletion mutants of 5 different US12 family members: US12 (3 of 4 donors), US14 (1 of 4 donors, with a trend towards increased NK activation in the other three donors), US18 (3 of 4 donors), US20 (4 of 4 donors), US21 (4 of 4 donors) (Figure 1B), while three US12 family deletion mutants (US15, US16, US19) reduced the level of NK cell activation in some donors (Figure 1B). Although members of the US12 family are capable individually of either activating or suppressing NK cell function, the net effect of the complete US12 gene family is clearly to inhibit NK cell recognition. Figure 1 Download asset Open asset Multiple US12 family proteins regulate NK activation. (A) Fibroblasts (HF-TERTs) were mock infected or infected with the Merlin strain of HCMV or the series of US12 family deletion mutants for 72 hr. Infected cells were incubated with donor PBMC for 5 hr and NK degranulation assessed by % CD107+ cells within the CD3-, CD56+ population by flow cytometry. (B) CD107 assay results (mean and SD) are shown from four separate donors performed in duplicate or triplicate and analysed by unpaired ordinary one way ANOVA with Dunnett’s test for multiple comparisons against the HCMV control *p<0.05, **p<0.01, ***p<0.005 ***p<0.001). Infected cells were assessed by the % cells with down-regulated MHC I compared to the mock-infected cells (for the experiment using donor #23 and # 385, HCMV 93%, ΔUS12 94%, ΔUS13 98%, ΔUS14 94%, ΔUS15 98%, ΔUS16 99%, ΔUS17 98%, ΔUS18 97%, ΔUS19 99%, ΔUS20 97%, ΔUS21 91%, ΔUS12-21 94%; for the experiment using donor #91 and # 285, HCMV 94%, ΔUS12 94%, ΔUS13 94%, ΔUS14 83%, ΔUS15 95%, ΔUS16 97%, ΔUS17 85%, ΔUS18 94%, ΔUS19 96%, ΔUS20 94%, ΔUS21 94%, ΔUS12-21 96%). https://doi.org/10.7554/eLife.22206.003 Table 1 HCMV constructs used in the study. https://doi.org/10.7554/eLife.22206.004 VirusBAC #Accession no.Cassette usedModificationPrevious referenceHCMV1111GU179001.1NoneRL13-, UL128-Stanton et al. (2010)△US121810KU221097GalKUS12 CDS deletedNone△US131831KU221099GalKUS13 CDS deletedNone△US141798KU221093GalKUS14 CDS deletedNone△US151800KU221094GalKUS15 CDS deletedNone△US161802KU221095GalKUS16 CDS deletedNone△US171804KU221096GalKUS17 CDS deletedNone△US181654KU221091RpsL-Neo-LacZUS18 CDS deletedFielding et al. (2014)△US191796KU221092RpsL-Neo-LacZUS19 CDS deletedNone△US201595KU221090SacB-AmpR-LacZUS20 CDS deletedFielding et al. (2014)△US211871KU221100RpsL-Neo-LacZUS21 CDS deletedNone△US12-211815KU221098RpsL-Neo-LacZUS12-21 deletedNone The US12 family targets plasma membrane proteins To provide an unbiased systematic analysis of the entire US12 gene family, we employed 10-plex tandem-mass tags (TMT) with MS3/Multinotch mass spectrometry to quantify whole cell (WCL) and plasma membrane (PM) proteomes in fibroblasts infected with the panel of HCMV US12 family mutants. The proteomic analyses were performed in two parts to permit inclusion of appropriate controls. Samples analyzed in Proteomic Series 1 included mock-infected controls, the parental HCMV strain, the US12-21 block deletion and defined mutants in US18, US19 and US20 (Figure 2, Figure 3, Figure 4A). Mass spectrometry quantified 7215 WCL and 1281 PM proteins. The extremely dynamic modulation of the host cell proteome observed during productive HCMV infection was consistent with previous findings (Figure 3)(Weekes et al., 2014). The role of the entire US12 gene family was assessed by comparison of the HCMV US12-21 block deletion mutant with its parental virus. The impact was most noticeably focused on the PM, in that the majority of WCL proteins affected were also PM ‘hits’ (Figure 2). The heat map illustrating changes in protein abundance correspondingly appears more dynamic i.e. a higher proportion of proteins detected were regulated in response to the deletion of individual US12 family genes for the PM proteome than for the much larger set of proteins quantified in WCL samples (Figure 3). For a given PM protein modulated by the US12 gene family (Figure 4), comparable results were observed in WCL samples (Figure 5), suggesting that the US12 family regulates protein expression or stability of cell surface proteins. Figure 2 Download asset Open asset The US12 family targets numerous plasma membrane proteins. Cells infected with HCMV (Merlin) or HCMV △US12-21 mutant were processed to give PM or WCL fractions and analyzed by TMT mass spectrometry. Scatter plot of proteins identified in the PM (panel A) or WCL fractions (panel B) respectively and quantified by 2 or more unique peptides. Fold change (△US12-21-infected fibroblasts/HCMV-infected cells) is shown as the log2 ratio on the x-axis and the signal:noise on the y-axis as log10. Proteins unaltered by the US21-21 deletion locate at the center of the plots (0 log2/1 fold-change), whereas proteins to the left or right of center represent proteins down- or up-regulated by the US12-21 deletion respectively. Significance B was used to estimate p values (Cox et al., 2009). The 2 different alleles of MICA present in HFs were detected by this analysis. https://doi.org/10.7554/eLife.22206.005 Figure 3 Download asset Open asset Hierarchical cluster analysis of all proteins quantified in proteomic series 1 and 2. Zoomed regions are shown for clusters of interest. https://doi.org/10.7554/eLife.22206.006 Figure 4 Download asset Open asset Proteomic analysis of cellular proteins targeted by US12 family members in PM samples. (A-B) Workflows of proteomic series 1 and 2 respectively. (C-E) Quantitation of PM proteins in enriched KEGG pathways identified using DAVID. Relative abundance of each protein is expressed relative to the sample with the highest abundance (set to 1). NQ – not quantified. A number of the US12-21 mutant targets were also regulated by the US18 and US20 mutants (B7-H6, ULBP2, IL6ST, KIT, KITLG JAM3, ACVR1, ACVRL1, IFNGR1, JAM3, MPZL1, CXADR, ALCAM, SDC4, CD99, SDC1). A subset of these proteins were also regulated by the US14 and/or US16 mutants (KIT, KITLG, ACVR1, IFNGR1, JAM3, MPZL1, CD99, SDC1). We estimated p values for the ratios of each mutant compared to HCMV Merlin using Benjamini-Hochberg corrected Significance B values (Cox and Mann, 2008): *p<0.05, **p<0.0001. For proteomic series 1, ratios were calculated as US12 family deletion mutant / average HCMV and for proteomic series 2, US12 family deletion mutant / HCMV. All proteins quantified by 2 or more peptides were included in this calculation. SDC1 was quantified by a single peptide in proteomic series 2. https://doi.org/10.7554/eLife.22206.007 Figure 5 Download asset Open asset Individual US12-family proteins target key natural killer cell ligands, cell adhesion molecules and cytokines and their receptors in WCL samples. (A–C) Quantitation of key US12 family targets in WCL samples (proteomic series 1 and 2 - comparative analysis of these proteins from PM samples is shown in Figure 3). We generally observed similar results for proteins quantified in both PM and WCL samples. Relative abundance of each protein is expressed relative to the sample with the highest abundance (set to 1). NQ – not quantified. P values were calculated as described in Figure 4: *p<0.05, **p<0.0001. MICB was only quantified by 1 peptide in proteomic series 1, and MICA, JAM3 and SDC4 by one peptide in proteomic series 2. https://doi.org/10.7554/eLife.22206.008 NK cells continually monitor the levels of inhibitory and activating ligands on the surface of potential targets, thus one of the roles of the US12 family in re-modeling the PM proteome is compatible with its role in directly impacting NK cell recognition. To gain an overview of pathways targeted, the online bioinformatics resource DAVID (Database for Annotation, Visualisation and Intergrated Discovery; https://david.ncifcrf.gov/) was used to perform a KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis on proteins rescued >3 fold by the deletion of the US12-21 block (Dennis et al., 2003; Huang et al., 2009). Multiple KEGG pathways were significantly enriched (data not shown), including natural killer cell-mediated cytotoxicity (MICA, MICB, ULBP2, IFNGR1), cytokine-cytokine receptor interaction (IL6ST, KIT, KITLG, ACVRL1, ACVR1, IFNGR1) and cell adhesion molecules (CAMs; JAM3, ICOSLG, PTPRM, MPZL1, CXADR, ALCAM, SDC4, CD99, SDC1). The modulation of cell adhesion molecules and chemokines/cytokines may additionally impact NK cell recognition. A further NK ligand, B7-H6, was not identified by DAVID analysis, but was also regulated by the US12-21 mutant. While MICA is a recognized target of US18 and US20 (Fielding et al., 2014), the regulation of the NK cell activating ligands B7H6, MICB and ULBP2 by the US12 family are novel. Functional independence and co-operation exerted by family members A substantial subset of the proteins regulated by the US12-21 block mutant were similarly modulated by US18 and US20 (Figure 4, Figure 5), whereas US19 specifically targeted RALGPS2 (Figure 6). To determine the contribution of each individual US12 family member to the overall effects observed with the ‘block’ deletion mutant, Proteomic Series two compared infection with all the single gene deletion mutants (except the US19 deletion) to the parent HCMV (Figure 4B). Mass spectrometry quantified 7156 whole cell and 1312 PM proteins. We re-examined key candidate molecules identified by bioinformatic analysis of the US12-21 block deletion (Figure 4C–E). A striking feature of this data was that the majority of these target proteins were regulated by both US18 and US20, with a subset additionally regulated to a lesser degree by US14 and/or US16 (Figure 4C–E). Other US12 family members also had important effects, with US12 and US13 mutants regulating the NKG2D ligands (NKG2DL) ULBP2 and MICB respectively, US14 mutant regulating PTPRM, and the US15 mutant regulating IL6ST (Figure 4C–E and Figure 6). Overall, the contribution made by a given family member varied dramatically ranging from the highly focused impact of US13 and US19 (one cellular target each) to the exceptionally promiscuous US20 (54 cellular targets) (Figure 6). Where a given protein was quantified both in PM and WCL samples, generally similar changes were observed in both samples (Figure 4 and 5). Interestingly, MICA was regulated by the deletion of US12-21 block to a much greater extent than any of the single deletion mutants, implying that multiple US12 family members may need to act in concert to optimize control over certain cellular targets (Figure 4C). Figure 6 Download asset Open asset Co-regulation of multiple cell surface proteins by >1 US12 family gene. Proteins that were rescued by deletion of each US12 family member in the PM fraction of proteomic Series 2 (i.e. not including US19) were analyzed to determine which were additionally targeted by one or more US12 family members. Proteins were plotted in a matrix with unique protein targets on the lower diagonal of the plot (US12 family member compared with itself) and common protein targets in intersections with other US12 members. To identify the highest confidence targets of the US12-21 family for the purposes of this analysis and to generate a shortlist of US12-21 family PM ‘hits’, we employed the following strategy: we included proteins (a) exhibiting at least 3-fold rescue upon deletion of a given US12 family member, quantified by at least two peptides and annotated by GO to indicate a PM location. (b) validated by a corresponding >2 fold change in at least one of (i) deletion of the US12-21 block (proteomic series 1 or 3), (ii) the biological repeat of US18 or US20 in proteomic series 1, (iii) the corresponding gene deletion in WCL proteomic series 2. This strategy identified all proteins shown in Figure 3. *US19 data was used from proteomic series 1. https://doi.org/10.7554/eLife.22206.009 Infection with the US21 deletion mutant resulted in selective impairment of many late phase Tp3 and Tp5 (Temporal protein profile 3 and 5) HCMV proteins (Weekes et al., 2014), for example UL32 and UL99/pp28, and elevated expression of pUS20, whereas the HCMV US12-21 block mutant did not recapitulate this effect (Figure 7, with further proteomic data searchable in Excel spreadsheet Supplemental file 1). Our data are consistent with deletion of the US21 structural gene impacting on the transcriptional control of US20 (data not shown), and may contribute to a potential growth defect observed for this particular mutant. Therefore the activation of NK cells in response to US21 mutant-infected targets cannot be assigned directly to an effect of US21 (Figure 1). A definitive assessment of the function of US21 will require an alternative approach. Figure 7 Download asset Open asset Altered expression of US20, UL32 and UL99 levels in the US21 deletion mutant-infected fibroblasts. Quantitation of US20, UL32 and UL99 in proteomic series 1–3 in both PM and WCL. Abundance of each protein is expressed relative to the sample with the highest abundance (set to 1). For proteomic series 1 and 2, p values were calculated as described in Figure 4: *p<0.05, **p<0.0001. US20 was only quantified by one peptide in proteomic series 1, WCL experiment. https://doi.org/10.7554/eLife.22206.010 Of all PM proteins regulated >3 fold by a member of the US12-21 family, 29% were regulated by 2 or more family members and 6% by 3 or more family members (Figure 6), although using a stringent 3-fold cutoff may underestimate the true incidence of co-regulation. Such co-regulation may suggest evolutionary pressure underpins the expansion of this gene family. Effect of lysosomal inhibition on US12 family targets The majority of proteins that the US12 family down regulates from the PM are also lost from the WCL, consistent with post-translational proteolysis or an overall reduction in expression. We previously found that US20 targets the NKG2DL MICA for degradation in lysosomes (Fielding et al., 2014). The DAVID pathway analysis identified increases in lysosomal proteins within the PM fraction of △US20-infected cells; the list of lysosomal proteins included a number of cathepsins and ATPases (PSAP, CTSD, ATP6V1E1, ATP6V1H, NPC2) (data not shown). These data suggest a role for US20 in regulating intracellular endo-lysosomal vesicular transport. To determine if this is a more general mechanism for the whole US12-21 family, HFFs were infected with HCMV, the US12-21 block deletion mutant or HCMV in the presence of the lysosomal protease inhibitor leupeptin for 12 hr prior to harvest, and protein expression was analyzed by 10-plex TMT at 24 hr, 48 hr and 72 hr post-infection (Proteomic Series 3, Figure 8A). Figure 8 Download asset Open asset Multiple US12 gene family targets are degraded via the lysosomal pathway. (A) Workflow of Proteomic Series 3. (B) Number of proteins targeted by the US12-21 block and additionally rescued >2 fold in the dataset by the △US12-21 deletion mutant or rescued >2 fold or >1.5 fold by leupeptin treatment for both WCL and PM. C. Comparable degree of rescue of US12-21 target proteins by US12-21 deletion and leupeptin treatment. Quantitation of a subset of these proteins is shown relative to the maximum abundance (set to 1). p-values were calculated as described in Figure 4, for the ratios of leupeptin treatment or US12-21 deletion virus infection at each time point compared to the matched wild-type Merlin-infected control. *p<0.05, **p<0.0001. SDC1 was only quantified by one peptide. https://doi.org/10.7554/eLife.22206.011 The △US12-21 ‘block’ deletion caused substantial shifts in WCL proteins, with >80 proteins rescued >2 fold at 48 hr, compared to the parent viral infection (Figure 8B). Of these, 26% were also rescued >2 fold and 51% > 1.5 fold with leupeptin suggesting that the US12 family targets multiple host proteins to the lysosome. Leupeptin treatment had a more limited effect on rescuing PM proteins back to the the cell surface (Figure 8B), consistent with our previous observation of intracellular accumulation of MICA without protein relocalisation to the cell surface during treatment with leupeptin or other lysosomal inhibitors (Fielding et al., 2014). Of the 21 proteins identified in Figure 4 within the key categories ‘natural killer cell-mediated cytotoxicity’, ‘cytokine-cytokine receptor interaction’ and ‘cell adhesion molecules’, 11 were rescued >2 fold by leupeptin, with the remainder exhibiting 1.3–2-fold rescue (Figure 8C). MICB was clearly rescued by leupeptin treatment (Figure 8C). Interestingly, control of MICB and ULBP2 levels correlated with regulation of UL16 by US13 and US12 (Figure 9). However, leupeptin rescue of ULBP2 and UL16 was less convincing than for MICB (Figure 9). Although many cellular substrates of the US12 family are clearly being targeted for lysosomal degradation, there may be additional mechanisms by which this family regulates the PM proteome. Figure 9 Download asset Open asset Regulation of UL16 levels by the US12 family. Quantitation of UL16 in proteomic series 1–3 in both PM and WCL. Relative abundance of each protein is expressed relative to the sample with the highest abundance (set to 1). For proteomic series 1 and 2, p values were calculated as described in Figure 4: *p<0.05, **p<0.0001. https://doi.org/10.7554/eLife.22206.012 Validation of PM protein regulation by US12 family members We used flow cytometry to validate a proportion of the various PM protein targets that are regulated by the US12 family (Figure 10). Next, we examined NKG2DL MICA and MICB (Figure 10). We found that MICA expression was rescued to a greater degree with the US12-21 block mutant than with the individual US18 or 20 deletion mutants (Figure 10). This is consistent with our previous analysis showing that a combined US18 and US20 deletion had a greater effect on cell surface MICA than single gene deletion mutants (Fielding et al., 2014). The US13 deletion mutant caused a minor, but consistent, elevation in cell surface expression of MICB. Other US12 family genes may yet contribute to regulating MICB, as there was a further increase in the US12-21 block mutant (Figures 10 and 4C). Figure 10 Download asset Open asset Validation of cell surface proteins regulated by the US12 family. Flow cytometry confirmed proteomic data for proteins representative of each category enriched in the DAVID analysis. Staining in mock/US12 family member deletion mutant infections (blue line) is shown relative to the parental HCMV infection (red line). Flow cytometry was carried out for cell surface expression of MHC I (W6/32) as a control for HCMC infection and isotype antibody staining controls (with directly PE-conjugated IgG1, IgG2a or IgG2b antibodies or for unconjugated antibodies mIgG and an anti-mouse-AF647 conjugated secondary antibody). Infected cells were assessed by the % cells with down-regulated MHC I compared to the mock-infected cells (HCMV 94%, ΔUS12 94%, ΔUS13 94%, ΔUS14 83%, ΔUS15 95%, ΔUS16 97%, ΔUS17 85%, ΔUS18 94%, ΔUS19 96%, ΔUS20 94%, ΔUS21 94%, ΔUS12-21 96%). Results are representative of at least two independent experiments. https://doi.org/10.7554/eLife.22206.013 B7-H6 is an NK cell activating ligand targeted by US18 and US20 The fact that, in addition to targeting MICA, US18 and US20 together are also implicated in targeting B7-H6 is extraordinary. B7-H6 is a known ligand for NKp30 and exogenous expression in fibroblasts increased NK degranulation (Figure 11A–C). Ectopic expression of US20, and to a lesser extent US18, from adenovirus vectors reduced levels of exogenously expressed B7-H6 detected by immunoblotting (Figure 11D). Both PM and WCL expression of B7-H6 was induced by the HCMV US18, US20 and US12-21 deletion mutants but not by the parental virus (Figure 4C, Figure 5A), which was confirmed by flow cytometry and immunoblot (Figures 10 and 12A). B7-H6 is therefore induced as a ‘stress ligand’ during productive infection, but its expression is controlled by US18 and US20, acting in concert, to target it for proteolysis (Figure 8C). Being the dominant ligand for the NK activating receptor NKp30, B7-H6 is potentially a critical target for HCMV. Figure 11 Download asset Open asset Adenovirus expressed B7-H6 regulates NK cell activation and B7-H6 levels are regulated by ectopically-expressed US18 and US20. (A) HF-CARs were infected with control (RAd-CTRL) or B7-H6-expressing (RAd-B7-H6) adenovirus vectors (MOI 5). Cells were harvested 48 h p.i. and used as targets in a CD107 degranulation assay with buffy-coat derived PBMC from 3 separate donors in duplicate or triplicate. Results (shown as mean and SD) were analyzed by unpaired two-tailed Student’s T-test. ***p<0.001, ****p<0.0001. (B and C

The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. In the 1990s, four chimeric viruses were constructed by replacing genome segments of the high passage Towne strain with segments of the low passage Toledo strain, with the goal of obtaining live attenuated vaccine candidates that remained safe but were more immunogenic than the overly attenuated Towne vaccine. The chimeras were found to be safe when administered to HCMV-seronegative human volunteers, but to differ significantly in their ability to induce seroconversion. This suggests that chimera-specific genetic differences impacted the ability to replicate or persist in vivo and the consequent ability to induce an antibody response. To identify specific genomic breakpoints between Towne and Toledo sequences and establish whether spontaneous mutations or rearrangements had occurred during construction of the chimeras, complete genome sequences were determined. No major deletions or rearrangements were observed, although a number of unanticipated mutations were identified. However, no clear association emerged between the genetic content of the chimeras and the reported levels of vaccine-induced HCMV-specific humoral or cellular immune responses, suggesting that multiple genetic determinants are likely to impact immunogenicity. In addition to revealing the genome organization of the four vaccine candidates, this study provided an opportunity to probe the genetics of HCMV attenuation in humans. The results may be valuable in the future design of safe live or replication-defective vaccines that optimize immunogenicity and efficacy.

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell-cell transmission. This process of cell-cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128-131A complex, a feature of WT but not passaged strains of HCMV.

ABSTRACT The effect of abrogating the interferon (IFN) response on human cytomegalovirus (HCMV) replication was investigated using primary human cells engineered to block either the production of or the response to type I IFNs. In IFN-deficient cells, HCMV produced larger plaques and spread and replicated more rapidly than in parental cells. These cells demonstrate the vital role of IFNs in controlling HCMV replication and provide useful tools to investigate the IFN response to HCMV.